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Titin kinase is an inactive pseudokinase scaffold that supports MuRF1 recruitment to the sarcomeric M-line.

Bogomolovas J, Gasch A, Simkovic F, Rigden DJ, Labeit S, Mayans O - Open Biol (2014)

Bottom Line: Inactivity is the result of two atypical residues in TK's active site, M34 and E147, that do not appear compatible with canonical kinase patterns.While not mediating stretch-dependent phospho-transfers, TK binds the E3 ubiquitin ligase MuRF1 that promotes sarcomeric ubiquitination in a stress-induced manner.Finally, we suggest that an evolutionary dichotomy of kinases/pseudokinases has occurred in TK-like kinases, where invertebrate members are active enzymes but vertebrate counterparts perform their signalling function as pseudokinase scaffolds.

View Article: PubMed Central - PubMed

Affiliation: Department of Integrative Pathophysiology, Medical Faculty Mannheim, University of Heidelberg, Mannheim 68167, Germany Institute of Integrative Biology, Biosciences Building, University of Liverpool, Crown St., Liverpool L69 7ZB, UK.

ABSTRACT
Striated muscle tissues undergo adaptive remodelling in response to mechanical load. This process involves the myofilament titin and, specifically, its kinase domain (TK; titin kinase) that translates mechanical signals into regulatory pathways of gene expression in the myofibril. TK mechanosensing appears mediated by a C-terminal regulatory tail (CRD) that sterically inhibits its active site. Allegedly, stretch-induced unfolding of this tail during muscle function releases TK inhibition and leads to its catalytic activation. However, the cellular pathway of TK is poorly understood and substrates proposed to date remain controversial. TK's best-established substrate is Tcap, a small structural protein of the Z-disc believed to link TK to myofibrillogenesis. Here, we show that TK is a pseudokinase with undetectable levels of catalysis and, therefore, that Tcap is not its substrate. Inactivity is the result of two atypical residues in TK's active site, M34 and E147, that do not appear compatible with canonical kinase patterns. While not mediating stretch-dependent phospho-transfers, TK binds the E3 ubiquitin ligase MuRF1 that promotes sarcomeric ubiquitination in a stress-induced manner. Given previous evidence of MuRF2 interaction, we propose that the cellular role of TK is to act as a conformationally regulated scaffold that functionally couples the ubiquitin ligases MuRF1 and MuRF2, thereby coordinating muscle-specific ubiquitination pathways and myofibril trophicity. Finally, we suggest that an evolutionary dichotomy of kinases/pseudokinases has occurred in TK-like kinases, where invertebrate members are active enzymes but vertebrate counterparts perform their signalling function as pseudokinase scaffolds.

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Dissection of TK/MuRF1 molecular interactions. (a) Identification of the MuRF1–titin interaction region by filter-binding assay. The helical domain of MuRF1 interacts with its established docking site A168–A170 and the C-terminally truncated version A168–A169, but not with the loop mutant A168–A170ΔKTLE, the N-terminally truncated A169–A170 or the single domain A169. Binding to A169–A170 is restored and enhanced when TK is included in the construct (A169-TK). The latter is used here in 10-fold lower quantity than the other samples. (b) Pull-down assay using skeletal muscle extract demonstrates that A168–A170 and A168-TK bind endogenous MuRF1 efficiently, but that the binding is stronger in the presence of TK. Neither GST nor TK alone are capable of pulling down endogenous MuRF1.
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RSOB140041F5: Dissection of TK/MuRF1 molecular interactions. (a) Identification of the MuRF1–titin interaction region by filter-binding assay. The helical domain of MuRF1 interacts with its established docking site A168–A170 and the C-terminally truncated version A168–A169, but not with the loop mutant A168–A170ΔKTLE, the N-terminally truncated A169–A170 or the single domain A169. Binding to A169–A170 is restored and enhanced when TK is included in the construct (A169-TK). The latter is used here in 10-fold lower quantity than the other samples. (b) Pull-down assay using skeletal muscle extract demonstrates that A168–A170 and A168-TK bind endogenous MuRF1 efficiently, but that the binding is stronger in the presence of TK. Neither GST nor TK alone are capable of pulling down endogenous MuRF1.

Mentions: Based on these findings, we reassessed how TK might contribute to muscle signalling in non-catalytic ways. The binding of the muscle-specific E3 ubiquitin ligase MuRF1 in the vicinity of TK is well documented [7,8]. MuRF1 binds a tandem of Ig-Ig-Fn domains, A168–A170, preceding the TK domain. The binding is mediated by the C-terminal helical domain of MuRF1 and determined by the presence of a loop with sequence KTLE in the titin domain A169 [8]. Here, we generated titin constructs comprising variations of the established MuRF1 docking site as well as TK (figure 5). A168–A170 and its C-terminally truncated variant A168A–A169 interacted with MuRF1, while the loop mutant A168–A170ΔKTLE (where the KTLE motif had been mutated to AAAA), N-terminally truncated A169–A170 and the single A169 domain did not display detectable interaction. Interestingly, when the fragment A169–A170 (which does not bind MuRF1 detectably) was extended to include TK, MuRF1 binding was restored and stronger than observed for the established A168–A170 locus (figure 5a). It is worth noting that the concentration of A169-TK was 10 times lower than that of the other constructs in the assay, indicating that TK markedly boost the MuRF1 interaction.Figure 5.


Titin kinase is an inactive pseudokinase scaffold that supports MuRF1 recruitment to the sarcomeric M-line.

Bogomolovas J, Gasch A, Simkovic F, Rigden DJ, Labeit S, Mayans O - Open Biol (2014)

Dissection of TK/MuRF1 molecular interactions. (a) Identification of the MuRF1–titin interaction region by filter-binding assay. The helical domain of MuRF1 interacts with its established docking site A168–A170 and the C-terminally truncated version A168–A169, but not with the loop mutant A168–A170ΔKTLE, the N-terminally truncated A169–A170 or the single domain A169. Binding to A169–A170 is restored and enhanced when TK is included in the construct (A169-TK). The latter is used here in 10-fold lower quantity than the other samples. (b) Pull-down assay using skeletal muscle extract demonstrates that A168–A170 and A168-TK bind endogenous MuRF1 efficiently, but that the binding is stronger in the presence of TK. Neither GST nor TK alone are capable of pulling down endogenous MuRF1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4042850&req=5

RSOB140041F5: Dissection of TK/MuRF1 molecular interactions. (a) Identification of the MuRF1–titin interaction region by filter-binding assay. The helical domain of MuRF1 interacts with its established docking site A168–A170 and the C-terminally truncated version A168–A169, but not with the loop mutant A168–A170ΔKTLE, the N-terminally truncated A169–A170 or the single domain A169. Binding to A169–A170 is restored and enhanced when TK is included in the construct (A169-TK). The latter is used here in 10-fold lower quantity than the other samples. (b) Pull-down assay using skeletal muscle extract demonstrates that A168–A170 and A168-TK bind endogenous MuRF1 efficiently, but that the binding is stronger in the presence of TK. Neither GST nor TK alone are capable of pulling down endogenous MuRF1.
Mentions: Based on these findings, we reassessed how TK might contribute to muscle signalling in non-catalytic ways. The binding of the muscle-specific E3 ubiquitin ligase MuRF1 in the vicinity of TK is well documented [7,8]. MuRF1 binds a tandem of Ig-Ig-Fn domains, A168–A170, preceding the TK domain. The binding is mediated by the C-terminal helical domain of MuRF1 and determined by the presence of a loop with sequence KTLE in the titin domain A169 [8]. Here, we generated titin constructs comprising variations of the established MuRF1 docking site as well as TK (figure 5). A168–A170 and its C-terminally truncated variant A168A–A169 interacted with MuRF1, while the loop mutant A168–A170ΔKTLE (where the KTLE motif had been mutated to AAAA), N-terminally truncated A169–A170 and the single A169 domain did not display detectable interaction. Interestingly, when the fragment A169–A170 (which does not bind MuRF1 detectably) was extended to include TK, MuRF1 binding was restored and stronger than observed for the established A168–A170 locus (figure 5a). It is worth noting that the concentration of A169-TK was 10 times lower than that of the other constructs in the assay, indicating that TK markedly boost the MuRF1 interaction.Figure 5.

Bottom Line: Inactivity is the result of two atypical residues in TK's active site, M34 and E147, that do not appear compatible with canonical kinase patterns.While not mediating stretch-dependent phospho-transfers, TK binds the E3 ubiquitin ligase MuRF1 that promotes sarcomeric ubiquitination in a stress-induced manner.Finally, we suggest that an evolutionary dichotomy of kinases/pseudokinases has occurred in TK-like kinases, where invertebrate members are active enzymes but vertebrate counterparts perform their signalling function as pseudokinase scaffolds.

View Article: PubMed Central - PubMed

Affiliation: Department of Integrative Pathophysiology, Medical Faculty Mannheim, University of Heidelberg, Mannheim 68167, Germany Institute of Integrative Biology, Biosciences Building, University of Liverpool, Crown St., Liverpool L69 7ZB, UK.

ABSTRACT
Striated muscle tissues undergo adaptive remodelling in response to mechanical load. This process involves the myofilament titin and, specifically, its kinase domain (TK; titin kinase) that translates mechanical signals into regulatory pathways of gene expression in the myofibril. TK mechanosensing appears mediated by a C-terminal regulatory tail (CRD) that sterically inhibits its active site. Allegedly, stretch-induced unfolding of this tail during muscle function releases TK inhibition and leads to its catalytic activation. However, the cellular pathway of TK is poorly understood and substrates proposed to date remain controversial. TK's best-established substrate is Tcap, a small structural protein of the Z-disc believed to link TK to myofibrillogenesis. Here, we show that TK is a pseudokinase with undetectable levels of catalysis and, therefore, that Tcap is not its substrate. Inactivity is the result of two atypical residues in TK's active site, M34 and E147, that do not appear compatible with canonical kinase patterns. While not mediating stretch-dependent phospho-transfers, TK binds the E3 ubiquitin ligase MuRF1 that promotes sarcomeric ubiquitination in a stress-induced manner. Given previous evidence of MuRF2 interaction, we propose that the cellular role of TK is to act as a conformationally regulated scaffold that functionally couples the ubiquitin ligases MuRF1 and MuRF2, thereby coordinating muscle-specific ubiquitination pathways and myofibril trophicity. Finally, we suggest that an evolutionary dichotomy of kinases/pseudokinases has occurred in TK-like kinases, where invertebrate members are active enzymes but vertebrate counterparts perform their signalling function as pseudokinase scaffolds.

Show MeSH
Related in: MedlinePlus